Overexpression of Calvin cycle enzyme fructose 1,6-bisphosphatase in Chlamydomonas reinhardtii has a detrimental effect on growth

Chlamydomonas reinhardtii and other green algae have potential for generating biofuels, pharmaceuticals, and other commercially valuable products. This potential could be improved by increasing the rate of growth and biomass accumulation. Overexpression of cyanobacterial fructose-1,6-bisphosphatase (cyanoFBPase-II), an enzyme that catalyzes an irreversible step in the Calvin cycle, improves growth and biomass accumulation in higher plants, so we set out to determine if overexpression of Chlamydomonas FBPase (CrFBPase) has the same effect in C. reinhardtii by using a chloroplast expression system. To this end, we generated a chloroplast expression vector that includes the C. reinhardtii FBP1 coding region synthesized with C. reinhardtii chloroplast codon-bias, flanked with psbD and psbA 5′ and 3′ regulatory sequences, respectively, and obtained transformants that integrated the vector properly into the chloroplast genome. CpFBP1 transformants accumulated increased levels of CrFBPase protein and extracts contained ~ 1.4-fold-increased levels of FBPase enzyme activity compared to the wild type strain. Surprisingly, we found that under photoautotrophic growth conditions and/or with elevated CO2 levels, this 40% elevation in FBPase activity had a negative effect overall on growth rate and biomass production. These results suggest that unlike the situation in higher plants, FBPase probably does not catalyze a rate-limiting step in the C. reinhardtii Calvin cycle.